Project Summary Bacillus anthracis (Ba) is a Gram-positive spore forming bacterium that is listed as an agent of highest concern (Category A) by NIAID and CDC. Ba is easy to grow, and its spores can be formulated into highly stable powder form and disseminated as aerosol or used to contaminate food or water. In 2001, letters laced with powdered anthrax spores were mailed to several US politicians. Twenty-two people, including 12 mail handlers, were infected, and five of them died. B. anthracis virulence largely depends on two key toxins generated by combination of the protective antigen (PA) associated with either lethal factor (LF) or edema factor (EF). Although some oral antibiotics and a vaccine are available for use, in practice these treatments cannot adequately address the adverse effects of bacterial toxins released post exposure. In our recently completed R41 project, we developed and tested a novel approach to target neutralizing anti-PA antibodies specifically to the site of infection in vitro and in vivo. The approach exploits the cell wall targeting domains (CWT) of well characterized phage endolysins (PlyG, PlyL and PlyB) that bind with species-specificity and high affinity to cell wall components of Ba. These CWTs are fused to specific antitoxin neutralizing monoclonal antibodies (mAbs) to generate Infection Site Targeted Antitoxin antibodies (ISTAbs). ISTAb technology provides two therapeutic advantages: immediate toxin neutralization at the site of infection preventing toxemia, and opsonophagocytic killing by phagocytes to simultaneously clear both bacteria and toxin. We compared nine ISTAb candidates (three CWTs and three mAbs) based on in vitro assays (cell binding and toxin neutralization) and selected one ISTAb (AVP-21D9-PlyG) for pre- and post-challenge in vivo studies in mice. This ISTAb exhibited significantly higher level of protection than the parental IgG. This R42 is aimed to take this lead ISTAb molecule into the next level in therapeutic pipeline. In this proposal, we will produce and extensively characterize next-generation AVP-21D9-PlyG ISTAbs, including stability and in vivo efficacy studies in mice and nonhuman primates (NHP), and develop a stable formulation. In Aim 1, we will use computer-aided optimizations to generate 3-5 ISTAb variants to remove potential liabilities that may complicate downstream development. In Aim 2, two lead candidates will be tested in mouse models. One lead molecule will be tested in an NHP model for PK and post-challenge efficacy. In Aim 3: The final ISTAb will be subjected to accelerated stability and PK studies, formulation, and generation of stable cell lines in CHO-S cells. The combination of immediate toxin clearance, phagocytic killing, and concurrent use of antibiotics, is expected to create synergy and yield a treatment that is far superior to the current standard of care. Furthermore, this technology can be applied to a variety of other bacterial pathogens where toxins play a key role in pathogenesis. Overall, this approach has board application as a platform technology across multiple pathogens.